CD4 and CD8 molecules play an important role in the identification of T lymphocytes, and diverse among fish species. In this study, CD4-1 and CD8α gene of spotted sea bass (Lateolabrax maculatus) were cloned, polyclonal antibodies against CD4-1 (CD4-1 pAbs) and CD8α (CD8α pAbs) were produced, respectively. And the variations in CD4-1 and CD8α T-lymphocytes in spotted sea bass and the cross-reactivity with leukocytes in pearl gentian grouper (Epinephelus fuscoguttatus x E. lanceolatus), schlegel's black rockfish (Sebastes schlegelii) and flounder (Paralichthys olivaceus) were investigated using CD4-1 pAbs and CD8α pAbs. The results showed that CD4-1 molecule ORF was 1413 bp and CD8α was 690 bp, both molecules are transmembrane glycoproteins with high amino acid homology to grouper. The CD4-1 pAbs specifically recognized both the CD4-1 recombinant and natural proteins, as does the CD8α pAbs to CD8α molecule, and no cross-reactivity between the two antibodies. CD4-1 and CD8α T lymphocytes were detected in peripheral blood, spleen and head kidney leukocytes in spotted sea bass. In cross-reactivity assay with other three fish, CD4-1 pAbs could recognize the lymphocytes from pearl gentian grouper and schlegel's black rockfish, both with highest proportions in the spleen leukocytes, 5.3 ± 0.4% and 2.6 ± 0.3%, respectively, and CD8α pAbs could only recognize the lymphocytes in pearl gentian grouper, and no cross-reactivities to lymphocytes of flounder. These data suggested that the CD4-1 and CD8α molecules varied by fish species in the genes features and antigenicity, which might result in the diversities of T lymphocytes subpopulations. This will be a key to elucidating the classification and evolution of T lymphocytes in fish.